Inducible gene expression systems based on antibiotics or hormones are a potent research tools and are constantly developed for their use in basic research and/or clinical application. Among the existing inducible transcriptional gene regulatory systems, the rtTA-regulatable system is the most widely exploited tool for controlling gene expression. This system, described for the first time in WO9429442, is based on a chimeric transcription factor consisting on the fusion of the bacterial tet repressor (TetR) with the activating domain of the viral protein 16 (VP-16), resulting in a tetracycline responsive transactivator (tTA). Random mutagenesis of tTA resulted in the rtTA (reverse tetracycline controlled trans-activator) protein that, contrary to tTA, requires the tetracycline to bind the tetO. The rtTA-based system requires the addition of tetracycline to activate transcription (TET-ON system) by allowing the binding of the rtTA to the TetO-CMV promoter. Several improvement of the rtTA have been done that improve inducibility and reduce background. However, all these tetracycline-inducible systems require a tetracycline-dependant-transactivator to activate the regulated promoter. The requirement of a transactivator for transcriptional activity has several undesired consequences, in particular, the promoter endogenous expression pattern may be altered due to the activation of the promoters of the regulated promoters, the possible activation of cellular genes due to the binding of the transactivator to pseudo-TetO sites and the toxicity caused by the presence of a transactivating domain makes these proteins very toxic. In fact, several studies have demonstrated that the rtTA-based systems can give rise to data misinterpretation due to the toxicity of the transactivator.
A doxycycline-regulated system based on the original TetR repressor was developed in 1998, by Yao and colleagues (Hum Gene Ther 1998; 9: 1939-50). The original tetR do not contain any transactivation domain and rely on blocking the activity of endogenous promoters. These characteristics should allow the design of a less-toxic Tet-inducible expression cassette that maintain the endogenous characteristics of the regulated-promoters and do not transactivate other cellular genes. Good results have been obtained using different vector systems for gene delivery (Nghiem P. et al., Proc Natl Acad Sci USA 2001; 98: 9092-7; Trapani J G and Korn S J., BMC Neurosci 2003; 4: 15; Reeves P J. et al., Proc. Natl. Acad. Sci. USA 2002; 99: 13419-24;
van de Wetering M. et al., EMBO Rep 2003; 4: 609-15 and Wiederschain D. et al. Cell Cycle 2009; 8: 498-504). However, most of these systems are based on two vector system and are reproducible only if the doxycycline-responsive cells are selected either by cloning or antibiotic selection. One of the reasons for this requirement is the high concentrations of TetR required to block promoter activity.
In spite of the potential advantages of the TetR system over the transactivator counterparts, the development of all-in-one regulatable vector systems based on TetR repressor has not been explored in detail. Wiederschain D et al. (Cell Cycle, 2009, 8: 498-504) have described a all-in-one vectors based on herpesvirus simplex (HSV). The use of HSV based vectors has been focused on neural cells due to their tropism, their toxicity and the difficulties to obtain high titre vectors.
Ogueta el al. have described an autoregulatable lentiviral vector without the requirement of antibiotic selection (Mol Med 2001; 7: 569-79). This autoregulatable vector express the TetR repressor through an internal ribosomal entry site (IRES) located downstream of the CMVTetO2 promoter. However, this systems has several potential drawbacks that could limit the use of the vector: 1—The TetR repressor and the regulated transgen are expressed through the CMVTetO promoter. Therefore, the steady-state TetR concentration required to block CMV expression will always allow expression of the transgene. 2—The efficiency of the IRES (from the EMCV) is cell-type specific and this can lead to the lost of doxycycline regulation in important target cells.
Accordingly, there is a need in the art for regulatable gene transfer vectors which overcome the disadvantages of the vectors known in the prior art.